Cells adhere to extracellular matrix (hereinafter abbreviated as ECM) mediated by a group of cell surface receptors which are termed integrins. Integrins perform their functions by forming 1:1 heterodimers of α and β chains. At least 18 types of α chain, 8 types of β chain and 24 types of αβ heterodimer have been identified and confirmed so far. It is known that each integrin recognizes a specific ligand. Integrins are classified into subfamilies depending upon their ligand specificities or functions, and divided into collagen receptors, laminin receptors, RGD receptors recognizing an Arg-Gly-Asp (RGD) sequence present in fibronectin, vitronectin, etc., and leukocyte-specific receptors present only in leukocytes (Hynes, R. O., 2002, Integrins: Bidirectional, Allosteric Signaling Machines. Cell 110: 673-87; Miyasaka, M., 2000, New edition of Adhesion Molecule Handbook, Shujunsya). The α4 and α9 integrins are members of a subfamily that does not belong to any of these types and called the α4 integrin subfamily (Elise L. Palmer, Curzio Rfiegg, Ronald Ferrando, Robert Pytela, Sheppard D., 1993, Sequence and Tissue Distribution of the Integrin α9 Subunit, a Novel Partner of β1 That Is Widely Distributed in Epithelia and Muscle. The Journal of Cell Biology, 123: 1289-97). Meanwhile, ECM used to be considered so far to serve as a mere cementing substance between cells. It has now become clear that the integrin-mediated ECM-cell interaction is significantly involved in regulating the growth, adhesion, movement, etc. of cells and associated with the onset of diseases including a progression of cancer, an exacerbation of inflammation, and the like.
For example, osteopontin (hereinafter abbreviated as OPN) which is one of the ECMs is a secreted, acidic phosphorylated glycoprotein with a molecular weight of about 41 kDa and is a molecule, whose expression is widely observed in breast milk, urine, renal tubules, osteoclasts, osteoblasts, macrophages, activated T cells, tumor tissues, and so forth. OPN has the adhesion sequences, GRGDS (SEQ ID NO:1) at the center of its molecule, the SVVYGLR (SEQ ID NO:2) sequence in human OPN or the SLAYGLR (SEQ ID NO:3) sequence in mouse OPN, and a thrombin-cleavage site in close proximity thereto, and binds through the GRGDS (SEQ ID NO:1) sequence to the RGD integrin or to the α4 (α4β1) and α9 (α9β1) integrins through the SVVYGLR (SEQ ID NO:2) sequence or the SLAYGLR (SEQ ID NO:3) sequence.
WO 02/081522 discloses a therapeutic effect on rheumatoid arthritis or hepatitis by inhibiting the OPN functions using OPN knockout mice or neutralizing antibodies against OPN. Moreover, this publication discloses that the SVVYGLR (SEQ ID NO:2) sequence is essential as recognizing the α9 and α4 integrins for pathogenesis of an inflammatory disease and that receptors for OPN are expressed in immunocompetent cells or the like and associated with an inflammatory disease.
Differences in binding profile have been found in that α4β1 binds both to OPN not cleaved with thrombin (uncleaved OPN) and to the N-terminal fragment of thrombin-cleaved OPN (cleaved OPN), whereas α9β1 binds only to the cleaved OPN (Y. Yokosaki, et al., (1999) The Journal of Biological Chemistry 274: 36328-36334; P. M. Green, et al., (2001) FEBS Letters, 503: 75-79; S. T. Barry, et al., (2000) Experimental Cell Research, 258: 342-351).
The α4 and α9 integrins share many common ligands other than OPN. Known ligands are the EDA domain of fibronectin, propeptide-von Willebrand factor (pp-vWF), tissue transglutaminase (tTG), blood coagulation factor XIII, vascular cell adhesion molecule-1 (VCAM-1), etc. In addition, the CS-1 domain of fibronectin, MadCAM-1 (α4β7), etc. are known as the ligands specifically recognized by the α4 integrin. Tenascin-C, plasmin, etc. are known as the ligands specifically recognized by the α9 integrin.
The amino acid sequences for the integrin subunits α9, α4 and β1 are publicly known. For instance, human α9 is registered as NM—002207, mouse α9 as NM—133721, human α4 as NM—000885, mouse α4 as NM—010576, human β1 as X07979, and mouse 131 as NM—010578, at the GenBank™. These integrins are also known to have high similarities between species in amino acid sequence.